Mitochondrial encephalomyopathies are important causes of cardiovascular disease, mental retardation and multisystem disease in humans. While individual causes of mitochondrial disease are rare, recent epidemiological evidence suggests that the minimal prevalence of respiratory chain mitochondrial diseases is 1 in 5000. Despite important recent insights into the clinical, biochemical, and molecular characterization of these disorders, specific genetic etiologies have been identified in only a minority of cases, and the underlying molecular pathogenesis remains poorly understood, with virtually no effective therapies available. The overall goal of this project is to provide new insights into mitochondrial biology and disease by performing a genetic screen in mouse embryonic stem (ES) cells that is designed to identify genes that are important for both normal mitochondrial function and are potential candidates for mitochondrial diseases. We have developed a means to use high throughput fluorescence activated cell sorting (FACS) to quantify parameters of mitochondrial function and have used a retroviral gene system to identify genes affecting mitochondrial function. We have demonstrated that mice can be generated from ES cells that carry mutations in genes known to cause human mitochondrial disease. Using this system, a novel method to perform a recessive genetic screen in mammalian ES cells is proposed. A traditional gene trap retroviral vector has been modified to contain an inducible system for antisense expression of the endogenous trapped locus. When induced, the resulting transcript is predicted to bind to the endogenous sense transcript, resulting in a long, double stranded RNA molecule that will activate endogenous RNAi pathways and will knockdown expression of the intact endogenous locus. In addition, an alternative RNAi screen using recently developed genome-wide shRNA lentivirus library will be employed. The development of these different approaches would represent an innovative means to perform unprecedented recessive genetic screens in ES cells. The identification of abnormal mitochondrial phenotypes in mutagenized mouse ES cells offers the promise of better understnading normal mitochondrial function, identifying candidate genes for human disease, generating new animal models for studying the pathophysiology of mitochondrial disease and delineating potential therapeutic targets. PUBLIC HEALTH RELEVANCE: Mitochondria are the structures found in cells that generate energy and regulate cell death. This proposal is to generate cell lines and animal models where specific genes that have some mitochondria functions are disrupted by novel genetic research tools.